Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.